1. Copyright Notice
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the xerographic reproduction by anyone of the patent document or the patent disclosure in exactly the form it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
2. Field of the Invention
This invention relates to the identification and characterization of microorganisms.
3. Background of the Invention
Multidrug resistance and human immunodeficiency virus (HIV-1) infections are factors which have had a profound impact on the tuberculosis problem. An increase in the frequency of Mycobacterium tuberculosis strains resistant to one or more anti-mycobacterial agents has been reported, Block, et al., (1994) JAMA 271:665-671. Immunocompromised HIV-1 infected patients not infected with M. tuberculosis are frequently infected with M. avium complex (MAC) or M. avium-M. intracellulare (MAI) complex. These mycobacteria species are often resistant to the drugs used to treat M. tuberculosis. These factors have re-emphasized the importance for the accurate determination of drug sensitivities and mycobacteria 6 species identification.
In HIV-1 infected patients, the correct diagnosis of the mycobacterial disease is essential since treatment of M. tuberculosis infections differs from that called for by other mycobacteria infections, Hoffner, S. E. (1994) Eur. J. Clin. Microbiol. Inf. Dis. 13:937-941. Non-tuberculosis mycobacteria commonly associated with HIV-1 infections include M. kansasii, M. xenopi, M. fortuitum, M. avium and M. intracellular, Wolinsky, E., (1992) Clin. Infect. Dis. 15:1-12, Shafer, R. W. and Sierra, M. F. 1992 Clin. Infect. Dis. 15:161-162. Additionally, 13% of new cases (HIV-1 infected and non-infected) of M. tuberculosis are resistant to one of the primary anti-tuberculosis drugs (isoniazid [INH], rifampin [RIF], streptomycin [STR], ethambutol [EMB] and pyrazinamide [PZA] and 3.2% are resistant to both RIF and INH, Block, et al., JAMA 271:665-671, (1994). Consequently, mycobacterial species identification and the determination of drug resistance have become central concerns during the diagnosis of mycobacterial diseases.
Methods used to detect, and to identify Mycobacterium species vary considerably. For detection of Mycobacterium tuberculosis, microscopic examination of acid-fast stained smears and cultures are still the methods of choice in most microbiological clinical laboratories. However, culture of clinical samples is hampered by the slow growth of mycobacteria. A mean time of four weeks is required before sufficient growth is obtained to enable detection and possible identification. Recently, two more rapid methods for culture have been developed involving a radiometric, Stager, C. E. et al., (1991) J. Clin. Microbiol. 29:154-157, and a biphasic (broth/agar) system Sewell, et al., (1993) J. Clin. Microbiol. 29:2689-2472. Once grown, cultured mycobacteria can be analyzed by lipid composition, the use of species specific antibodies, species specific DNA or RNA probes and PCR-based sequence analysis of 16S rRNA gene (Schirm, et al. (1995) J. Clin. Microbiol. 33:3221-3224; Kox, et al. (1995) J. Clin. Microbiol. 33:3225-3233) and IS6110 specific repetitive sequence analysis (For a review see, e.g., Small et al., P. M. and van Embden, J. D. A. (1994) Am. Society for Microbiology, pp. 569-582). The analysis of 16S rRNA sequences (RNA and DNA) has been the most informative molecular approach to identify Mycobacteria species (Jonas, et al., J. Clin. Microbiol. 31:2410-2416 (1993)). However, to obtain drug sensitivity information for the same isolate, additional protocols (culture) or alternative gene analysis is necessary.
To determine drug sensitivity information, culture methods are still the protocols of choice. Mycobacteria are judged to be resistant to particular drugs by use of either the standard proportional plate method or minimal inhibitory concentration (MIC) method. However, given the inherent lengthy times required by culture methods, approaches to determine drug sensitivity based on molecular genetics have been recently developed.
Table 1 lists the M. tuberculosis genes with which when mutated have been shown to confer drug resistance (other genes are known, e.g., the pncA gene). Of the drugs listed in Table 1, RIF and INH form the backbone of tuberculosis treatment. Detection of RIF resistance in M. tuberculosis is important not only because of its clinical and epidemiological implications but also because it is a marker for the highly threatening multidrug resistant phenotype (Telenti, et al. (1993) The Lancet 341:647-650). Of the drug resistances listed in Table 1, decreased sensitivity to RIF is the best understood on a genetic basis.
TABLE 1M. tuberculosis Genes with MutationsWhich Confer Drug ResistanceDrugGeneSize (bp)Gene ProductRIFrpoB3,534β-subunit of RNA polymeraseINHkatG2,205catalase-peroxidaseINH-ETHinhA810fatty and biosynthesisSTRrpsL372ribosomal protein S12rrs1,46416S rRNAFQgyrA2,517DMA gyrase A subunit
Because resistance to RIF in E. coli strains was observed to arise as a result of mutations in the rpoB gene, Telenti, et al., id., identified a 69 base pair (bp) region of the M. tuberculosis rpoB gene as the locus where RIF resistant mutations were focused. Kapur, et al., (1995) Arch. Pathol. Lab. Med. 119:131-138, identified additional novel mutations in the M. tuberculosis rpoB gene which extended this core region to 81 bp. In a detailed review on antimicrobial agent resistance in mycobacteria, Musser (Clin. Microbiol. Rev., 8:496-514 (1995)), summarized all the characterized mutations and their relative frequency of occurrence in this 81 bp region of rpoB. Missense mutations comprise 88% of all known mutations while insertions (3 or 6 bp) and deletions (3, 6 and 9 bp) account for 4% and 8% of the remaining mutations, respectively. Approximately 90% of all RIF resistant tuberculosis isolates have been shown to have mutations in this 81 bp region. The remaining 10% are thought possibly to involve genes other than rpoB.
For the above reasons, it would be desirable to have simpler methods which identify and characterize microorganisms, such as Mycobacteria, both at the phenotypic and genotypic level. This invention fulfills that and related needs.